Factor VIII:C procoagulant protein (also known as "Antihemophilic Factor") is a participant in the intrinsic pathway of blood coagulation, acting as a cofactor in the activation of factor X. Factor VIII:C procoagulant protein (factor VIII:C) circulates at low concentration (about 200 ng/ml) in the plasma as a non-covalently linked complex with von Willebrand factor (vWF).
Several hereditary disorders are associated with these two proteins. In individuals with the hereditary X chromosome-linked bleeding disorder, hemophilia A (also known as "classic hemophilia"), factor VIII:C activity is absent. Hemophilia A is the most common hereditary disorder of coagulation, affecting about six men in every 100,000 (Bloom, Nature 303:474-475, 1983). Von Willebrand's disease is a hereditary bleeding disorder which results in extended bleeding time due to reduced levels of active vWF. This disorder affects about one person in every 100,000 (L. Harke, Hemostatis Manual, 2d ed., F. A. Davis Co., Philadelphia, Pa., 1974). Currently, individuals affected by these two bleeding disorders are treated with concentrates rich in factor VIII:C and vWF. These protein concentrates, prepared from the pooled blood of a large number of donors, are expensive to produce and, though enriched for the specific factors required, still contain less than 1% factor VIII:C and are contaminated with other proteins. In addition, there is a risk of viral contamination (e.g., hepatitis viruses and HIV-I) in the concentrates due to the use of pooled human plasma as the source of these coagulation factors and many hemophiliacs receiving the concentrates have been infected.
Purification of factor VIII:C has been complicated by its low abundance in plasma, extreme lability, and association with von Willebrand factor. Although expression of cloned factor VIII:C in recombinant cells has been achieved (Wood et al., Nature 312:330-337, 1984; Toole et al., Nature 312:342-347, 1984; Truett et al., DNA 4:333-349, 1985), the recombinant proteins have not been extensively purified or characterized.
A number of purification methods for factor VIII:C have been attempted, although they are characterized by somewhat limited efficiency. For instance, Farrugia et al. (Thromb. Haemost. 51:338-342, 1984) described a factor VIII:C purification method which involved precipitation of factor VIII:C from plasma or cryoprecipitate with hydrophilic polymers, while Wagner et al. (Thromb. Diath. Haemorrh. 11:64, 1964) described the purification of factor VIII:C from plasma or cryoprecipitate using precipitation with lecithins. In addition, Madaras et al. (Haemost. 7:321-331, 1978) have described the purification of factor VIII:C from plasma or cryoprecipitate using chromatography on ion-exchange columns and gel filtration followed by heparin-sepharose affinity chromatography. Knutson and Fass (Blood 59:615-624, 1982) described a multistep process for the purification of porcine factor VIII:C. In general, these methods produce factor VIII:C in low yields and, in many cases, as a complex with vWF.
The preparation of highly purified bovine factor VIII:C derived from bovine plasma has been described by Vehar and Davie (Biochemistry 19:401-410, 1980) using gel filtration and chromatography on a factor X-sepharose column as the final step of the purification procedure. Tuddenham et al. (J. Lab. Clin. Med. 93:40-53, 1979) have described a method for purifying factor VIII:C using immunoaffinity chromatography. This method utilizes polyclonal antisera directed against vWF to adsorb the factor VIII:C-vWF complex from plasma. Purified factor VIII:C is eluted from the column using a calcium ion gradient. Austen (British J. Haemat. 43:669, 1979) described a method for separating factor VIII:C from contaminating plasma proteins using aminohexyl sepharose chromatography. These methods do not, however, result in a concentrated product.
Zimmerman and Fulcher (U.S. Pat. No. 4,361,509; 1982) have disclosed a method for preparing a concentrated high-purity factor VIII:C using a two-column method. The first column, consisting of monoclonal antibodies directed against vWF bound to agarose beads, served to purify factor VIII:C from the starting material. The second column, used for concentration of the purified factor VIII:C, consisted of aminohexyl-substituted agarose.
The major disadvantages posed by the use of immunoaffinity chromatography, as described by Tuddenham et al. (ibid.) and Zimmerman and Fulcher (ibid.), are in the regeneration and reusability of the affinity matrix and the contamination of the product with nonhuman antibodies. Immunoaffinity columns, as described above, bind the vWF portion of the factor VIII:C-vWF complex. Calcium ion treatment of the immunoaffinity column releases free factor VIII:C, but the vWF remains tightly bound to the column. However, the factor VIII:C purified by this method is often contaminated with nonhuman antibodies that have been leached from the column. Further, the tight bond between antibody and vWF necessitates the use of powerful desorption agents to achieve elution of the bound vWF as a prelude to reuse of the column. These procedures can lead to loss of the biological activity of the vWF and/or loss of the immunological properties of the antibody, resulting in a short-lived column. Use of such columns for the commercial preparation of factor VIII:C is therefore expensive, while their use for the isolation of biologically active vWF is difficult at best. Additionally, the use of strong desorption agents, some of which are toxic to human systems, requires their removal from the product before use.
In view of the disadvantages of current methods employed for purifying factor VIII:C and vWF, there is a need in the art for an alternative purification method which provides a high yield of pure factor VIII:C, vWf and/or the factor VIII:C-vWF complex. The present invention fulfills this need, and further provides other related advantages.